The mechanism(s) of nanoparticle–cell interactions are still not understood. At present there is little knowledge of the relevant length‐ and timescales for nanoparticle intracellular entry and ...localization within cells, or the cell‐specificity of nanoparticle uptake and localisation. Here, the effect of particle size on the in‐vitro intracellular uptake of model fluorescent carboxyl‐modified polystyrene nanoparticles is investigated in various cell lines. A range of micro‐ and nanoparticles of defined sizes (40 nm to 2 μm) are incubated with a series of cell types, including HeLa and A549 epithelial cells, 1321N1 astrocytes, HCMEC D3 endothelial cells, and murine RAW 264.7 macrophages. Techniques such as confocal microscopy and flow cytometry are used to study particle uptake and subcellular localisation, making significant efforts to ensure reproducibility in a semiquantitative approach. The results indicate that internalization of (nano)particles is highly size‐dependent for all cell lines studied, and the kinetics of uptake for the same type of nanoparticle varies in the different cell types. Interestingly, even cells not specialized for phagocytosis are able to internalize the larger nanoparticles. Intracellular uptake of all sizes of particles is observed to be highest in RAW 264.7 cells (a specialized phagocytic cell line) and the lowest in the HeLa cells. These results suggest that (nano)particle uptake might not follow commonly defined size limits for uptake processes, and highlight the variability of uptake kinetics for the same material in different cell types. These conclusions have important implications for the assessment of the safety of nanomaterials and for the potential biomedical applications of nanoparticles.
Uptake and localization of Texas Red‐labeled, carboxylate‐modified polystyrene nanoparticles are studied comparatively across a panel of cell lines. Internalization of the particles is found to be highly size‐dependent for all cell lines studied, but the uptake kinetics vary across different cell types. Intracellular uptake is found to be highest in RAW 264.7 macrophages and lowest in HeLa cells. The results suggest that (nano)particle uptake may not follow the size limits commonly defined for uptake processes, and highlight the variability of uptake kinetics for the same material in different cell lines, with important implications regarding the safety of nanomaterials and their use in biomedical applications.
The human microbiome plays an important role in cancer. Accumulating evidence indicates that commensal microbiome-derived DNA may be represented in minute quantities in the cell-free DNA of human ...blood and could possibly be harnessed as a new cancer biomarker. However, there has been limited use of rigorous experimental controls to account for contamination, which invariably affects low-biomass microbiome studies.
We apply a combination of 16S-rRNA-gene sequencing and droplet digital PCR to determine if the specific detection of cell-free microbial DNA (cfmDNA) is possible in metastatic melanoma patients. Compared to matched stool and saliva samples, the absolute concentration of cfmDNA is low but significantly above the levels detected from negative controls. The microbial community of plasma is strongly influenced by laboratory and reagent contaminants introduced during the DNA extraction and sequencing processes. Through the application of an in silico decontamination strategy including the filtering of amplicon sequence variants (ASVs) with batch dependent abundances and those with a higher prevalence in negative controls, we identify known gut commensal bacteria, such as Faecalibacterium, Bacteroides and Ruminococcus, and also other uncharacterised ASVs. We analyse additional plasma samples, highlighting the potential of this framework to identify differences in cfmDNA between healthy and cancer patients.
Together, these observations indicate that plasma can harbour a low yet detectable level of cfmDNA. The results highlight the importance of accounting for contamination and provide an analytical decontamination framework to allow the accurate detection of cfmDNA for future biomarker studies in cancer and other diseases.
The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create ...functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.
Abstract Ultrasmall nanoparticulate materials with core sizes in the 1-3 nm range bridge the gap between single molecules and classical, larger-sized nanomaterials, not only in terms of spatial ...dimension, but also as regards physicochemical and pharmacokinetic properties. Due to these unique properties, ultrasmall nanoparticles appear to be promising materials for nanomedicinal applications. This review overviews the different synthetic methods of inorganic ultrasmall nanoparticles as well as their properties, characterization, surface modification and toxicity. We moreover summarize the current state of knowledge regarding pharmacokinetics, biodistribution and targeting of nanoscale materials. Aside from addressing the issue of biomolecular corona formation and elaborating on the interactions of ultrasmall nanoparticles with individual cells, we discuss the potential diagnostic, therapeutic and theranostic applications of ultrasmall nanoparticles in the emerging field of nanomedicine in the final part of this review.
Geological identification of past tsunamis is important for risk assessment studies, especially in areas where the historical record is limited or absent. The main problem when using the geological ...evidence is to distinguish between tsunami and storm deposits. Both are high-energy events that may leave marine traces in coastal stratigraphic sequences. At Martinhal, SW Portugal both storm surge and tsunami deposits are present at the same site within a single stratigraphic sequence, which makes it suitable to study the differences between them, excluding variations caused by local factors.
The tsunami associated with the Lisbon earthquake of November 1st 1755 AD, had a major impact on the geomorphology and sedimentology of Martinhal. It breached the barrier and laid down an extensive sheet of sand, as described in eyewitness reports. Besides the tsunami deposit the stratigraphy of Martinhal also displays evidence for storm surges that have breached and overtopped the barrier, flooding the lowland and leaving sand layers. Both marine-derived flood deposits show similar grain size characteristics and distinctive marine foraminifera. The most important differences are the rip-up clasts and boulders exclusively found in the tsunami deposit and the landward extent of the tsunami deposit that everywhere exceeds that of the storm deposits. Identification of both depositional units was only possible using a collection of different data and extensive stratigraphical information from cores as well as trenches.
The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond ...metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasis and after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.
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•scRNA-seq of the healthy and injured liver epithelium reveals dynamic heterogeneity•Transcriptional heterogeneity is driven by fluctuating activation of YAP signaling•YAP is essential for adult biliary cell survival under homeostatic conditions•YAP is required in hepatocytes for the ductular response during regeneration
The transcriptional landscape of the epithelium in healthy and regenerating murine livers was investigated, revealing a dynamically fluctuating and heterogeneous YAP transcriptional program. Further analysis uncovered YAP signaling dualism: it is essential in biliary epithelial cells for homeostatic maintenance and in hepatocytes for the regenerative response to injury.
A wealth of genomic and epigenomic data has identified abnormal regulation of epigenetic processes as a prominent theme in hematologic malignancies. Recurrent somatic alterations in myeloid ...malignancies of key proteins involved in DNA methylation, post-translational histone modification and chromatin remodeling have highlighted the importance of epigenetic regulation of gene expression in the initiation and maintenance of various malignancies. The rational use of targeted epigenetic therapies requires a thorough understanding of the underlying mechanisms of malignant transformation driven by aberrant epigenetic regulators. In this review we provide an overview of the major protagonists in epigenetic regulation, their aberrant role in myeloid malignancies, prognostic significance and potential for therapeutic targeting.
Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach ...to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.
The fast-paced development of nanotechnology needs the support of effective safety testing. We have developed a screening platform measuring simultaneously several cellular parameters for exposure to ...various concentrations of nanoparticles (NPs). Cell lines representative of different organ cell types, including lung, endothelium, liver, kidney, macrophages, glia, and neuronal cells were exposed to 50 nm amine-modified polystyrene (PS-NH2) NPs previously reported to induce apoptosis and to 50 nm sulphonated and carboxyl-modified polystyrene NPs that were reported to be silent. All cell lines apart from Raw 264.7 executed apoptosis in response to PS-NH2 NPs, showing specific sequences of EC50 thresholds; lysosomal acidification was the most sensitive parameter. Loss of mitochondrial membrane potential and plasma membrane integrity measured by High Content Analysis resulted comparably sensitive to the equivalent OECD-recommended assays, allowing increased output. Analysis of the acidic compartments revealed good cerrelation between size/fluorescence intensity and dose of PS-NH2 NPs applied; moreover steatosis and phospholipidosis were observed, consistent with the lysosomal alterations revealed by Lysotracker green; similar responses were observed when comparing astrocytoma cells with primary astrocytes. We have established a platform providing mechanistic insights on the response to exposure to nanoparticles. Such platform holds great potential for in vitro screening of nanomaterials in highthroughput format.
It is shown that a global atmospheric model with horizontal resolution typical of that used in operational numerical weather prediction is able to simulate non‐gaussian probability distributions ...associated with the climatology of quasi‐persistent Euro‐Atlantic weather regimes. The spatial patterns of these simulated regimes are remarkably accurate. By contrast, the same model, integrated at a resolution more typical of current climate models, shows no statistically significant evidence of such non‐gaussian regime structures, and the spatial structure of the corresponding clusters are not accurate. Hence, whilst studies typically show incremental improvements in first and second moments of climatological distributions of the large‐scale flow with increasing model resolution, here a real step change in the higher‐order moments is found. It is argued that these results have profound implications for the ability of high resolution limited‐area models, forced by low resolution global models, to simulate reliably, regional climate change signals.
Key Points
Regimes not represented in an atmosphere model at typical climate resolution
Skillful representation of regimes at higher weather model resolution
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